A living body is protected from foreign substances mainly by an immune response, and an immune system has been established by various cells and the soluble factors produced thereby. Among them, leukocytes, especially lymphocytes, play a key role. The lymphocytes are classified in two major types, B lymphocyte (hereinafter referred to as B cell) and T lymphocyte (hereinafter referred to as T cell), both of which specifically recognize an antigen and act on the antigen to protect the living body.
T cell is subclassified to helper T cell having CD(Cluster Designation)4 marker (hereinafter referred to as TH), mainly involved in assisting in antibody production and induction of various immune responses, and cytotoxic T cell having CD8 marker (Tc: cytotoxic T lymphocyte, also referred to as killer T cell, which may be hereinafter referred to as CTL), mainly exhibiting a cytotoxic activity. CTL, which plays the most important role in recognizing, destroying and eliminating tumor cell, virus-infected cell or the like, does not produce an antibody specifically reacting with an antigen like in B cell, but directly recognizes and acts on antigens (antigenic peptide) from a target cell which is associated with major histocompatibility complex (MHC, which may be also referred to as human leukocyte antigen (HLA) in human) Class I molecules present on the surface of the target cell membrane. At this time, T cell receptor (hereinafter referred to as TCR) existing on the surface of the CTL membrane specifically recognizes the above-mentioned antigenic peptides and MHC Class I molecules, and determines whether the antigenic peptide is derived from itself or nonself. Target cell which has been determined to be from nonself is then specifically destroyed and eliminated by CTL.
Recent years, a therapy which would cause a heavier physical burden on a patient, such as pharmacotherapy and radiotherapy, has been reconsidered, and an interest has increased in an immunotherapy with a lighter physical burden on a patient. Especially, there has been remarked an effectiveness of adoptive immunotherapy in which CTL capable of specifically reacting with an antigen of interest is induced in vitro from CTL or T cell derived from a human having normal immune function, and then transferred to a patient. For instance, it has been suggested that adoptive immunotherapy using an animal model is an effective therapy for virus infection and tumor (authored by Greenberg, P. D., Advances in Immunology, published in 1992). Further, use of CTL to a patient with congenital, acquired or iatrogenic T cell immunodeficiency has been remarked, from the fact that administration of CIL to a patient with immunodeficiency results in reconstruction of specific CIL response, by which cytomegalovirus is rapidly and persistently eliminated without showing toxicity [Blood, 78, 1373-1380 (1991)] and the like. In this therapy, it is important to maintain or increase the cell count with maintaining or enhancing the antigen-specific cytotoxic activity of the CTL. Also, as to maintenance and increase of the cell count of CTL, if an effective cell count in adoptive immunotherapy for human is deduced on the basis of the studies on an animal model, it is thought that 109 to 1010 antigen-specific T cells are necessary (authored by Greenberg, P. D., Advances in Immunology, published in 1992). In other words, in adoptive immunotherapy, it can be said that it is a major problem to obtain the above cell count in vitro in a short period of time.
As to maintenance and enhancement of an antigen-specific cytotoxic activity of CTL, there has been generally employed a method of repeating stimulation with an antigen of interest when a specific response to an antigen for CTL is induced. However, in this method, the cell count may temporarily be increased, but the cell count is eventually decreased, and necessary cell count cannot be obtained. As its countermeasure, there are no other means in the current situation but to lyophilize the cells in an earlier stage during repeat of stimulation with an antigen, or to obtain antigen-specific CTL clones, lyophilize a part of the clones, and repeat antigen stimulation to the lyophilized cells after thawing if the cell count or antigen-specific cytotoxic activity of the CTL clones is lowered due to a long-term culture.
A method for establishing T cell by a long-term culture using mouse T cell has been reported [Nature, 294, 697-699 (1981)], which is a method for isolating T cell and establishing a cell strain therewith. However, it is impossible to proliferate T cell to 109 to 1010 cells by this method. Next, U.S. Pat. No. 5,057,423 discloses a method comprising inducing lymphokine-activated killer (LAK) cell using a large amount of interleukin 2 (IL-2) in a high concentration, thereby increasing the cell count in 100 folds in 3 to 4 days. This cell count is enormous, considering that it usually takes about 24 hours for a single cell to be divided and proliferated into two cells. In addition, adoptive immunotherapy has been tried by inducing tumor-infiltrating lymphocyte (TIL) using IL-2 in a high concentration as above [New Engl. J. Med., 316, 1310-1321 (1986); New Engl. J. Med., 319, 1676-1680 (1988); Blood, 81, 2093-2101 (1993)]. However, the former is a method for obtaining T cell which is non-specific for an antigen, and in the latter, antigen specificity is very low, if any, because activated polyclonal lymphocyte population is used. Further, in both of the above-mentioned methods, IL-2 is used in a high concentration in order to promote cell proliferation. It is reported that apoptosis (cell death) may occur when T cell treated with IL-2 in a high concentration is stimulated with a specific antigen in the absence of IL-2 [Nature, 353, 858-861 (1991); Eur. J. Immunol., 23, 1552-1560 (1992)]. Therefore, the effectiveness of LAK cell or TIL obtained by the above-mentioned methods is problematic.
In addition, when T cell is cultured at a low density (5×103 to 1×104 cells/ml) in the presence of T-cell growth factor and IL-2, T cell rapidly proliferates over a period of 7 days, and eventually proliferates to a saturation density of 3 to 5×105 cells/ml. However, it is also reported that the cell always dies once the cell reaches the saturation density [Immunological Rev., 54, 81-109 (1981)]. Therefore, LAK cell, TIL and the method for culturing T cell at a low density are problematic in both aspects of actual use and usefulness.
Next, regarding the antigen-specific CTL, there are reported adoptive immunotherapy in which allogenic cytomegalovirus(CMV)-specific CTL is cultured in vitro for 5 to 12 weeks to proliferate CTL, and then administered intravenously to a patient with immunodeficiency [Riddell et al., Science, 257:238-240, 1992]; and a method for isolating and expanding a CMV-specific CTL clone using self-CMV infected fibroblast and IL-2 [J. Immunology, 146, 2795-2804 (1991)] or using anti-CD3 monoclonal antibody (anti-CD3 mAb) and IL-2 [J. Imm. Methods, 128, 189-201 (1990)]. However, there is a serious problem in these methods. Specifically, it takes about 3 months to obtain 1×109 cells/ml of antigen-specific CTLS, during which time the symptoms of the patient advance, so that it is difficult to appropriately treat the disease depending on the situation.
As a method of solving the above-mentioned problem, WO 96/06929 discloses an REM method (rapid expansion method). This REM method is a method for expanding a primary T cell population containing antigen-specific CTL and TH in a short period of time. In other words, this method is characterized in that a large amount of T cell can be provided by expanding individual T cell clones. However, there is a problem as described below. In the REM method, antigen-specific CTL is expanded using anti-CD3 antibody, IL-2, and PBMC (peripheral blood monocyte) made deficient in an ability for proliferation by irradiation, and Epstein-Barr virus (EBV)-infected cells. However, there are problems that risk of admixing EBV-transformed B cell (EBV-B cell) into T cell is not deniable (problem in safety); that a large amount of PBMC (PBMC in an amount of about 40 times the number of antigen-specific CTL required) is required as feeder cell; that the antigen-specific cytotoxic activity of the expanded CTL cannot be sufficiently satisfactory; that the antigen-specific cytotoxic activity possessed by T cell is decreased with the cell proliferation when CTL is allowed to proliferate using a T cell population other than the T cell clone; and the like.
In other words, in a conventional method for preparing antigen-specific CTL, there have not been solved the problems essential to adoptive immunotherapy in which CTL having an antigen-specific cytotoxic activity effectively used in the treatment, is prepared in a sufficient amount for a short period of time.
An object of the present invention is to provide methods for inducing, maintaining and expanding CTL having an antigen-specific cytotoxic activity at a high level, which is suitably used in adoptive immunotherapy.